Project Summary This proposal investigates structural and functional mechanisms of two related receptors from the family of protein kinases, RNase L (Aims 1, 2) and Gcn2 (Aim 3). These receptors are ubiquitously expressed in human tissues and regulate protein synthesis (translation) to mitigate cellular stresses. RNase L senses stress caused by double-stranded RNA (dsRNA), whereas Gcn2 detects stress caused by nutrient limitation. Mammalian cells are highly sensitive to the presence of dsRNA. DsRNA molecules are normally rare, but are widespread in virus-infected cells and in cancer cells due to upsurge of dsRNA-rich endogenous repeat elements, which makes studying dsRNA responses critically important. Yet, we have a poor understanding of the dsRNA-RNase L pathway. Our goal in Aims 1 and 2 of this proposal is to understand the molecular mechanism regulating RNase L generally and to extensively characterize the novel mechanism of translational regulation by RNase L, discovered by our laboratory. This work is biomedically significant because RNase L, in addition to having anticancer and antiviral roles, is an antilipogenic receptor that could be important for treating and preventing infectious, neoplastic, and metabolic illnesses. In Aim 1 we will elucidate the mechanism of RNase L regulation by obtaining the structure of RNase L in its latent state. Our hypothesis is that RNase L forms a defined latent structure that restricts uncontrollable RNase L signaling to protect healthy tissues. We will test this hypothesis using structural analysis by cryo electron microscopy (cryo-EM). In Aim 2, we will use cell biology, biochemistry and an innovative RNA-seq approach developed in our group to establish the mechanism of translation regulation by RNase L. In 2019, we published our discovery that RNase L performs endonucleolytic cleavage of actively translating mRNAs as a strategy to control cell-wide protein synthesis. Preliminary data in this proposal provide further insights into this mRNA decay and show that RNase L exhibits a distinctive preference for mRNA coding regions, leading us to the hypothesis that the action of RNase L is coupled to translation. We will test this hypothesis thoroughly in our investigation. In Aim 3, we extend our work to a related receptor regulating global translation, Gcn2. Gcn2 is a mechanistically poorly understood stress kinase located proximally to RNase L in the human kinome. Gcn2 is important for proteostasis, memory function, and cancer metabolism. Gcn2 serves as a sensor of starvation that, upon binding uncharged tRNAs, inhibits global protein synthesis to mitigate nutrient deficits. The mechanism of Gcn2 activation is poorly understood due to the absence of structural information about its regulatory tRNA-sensing domain. We will employ cryo-EM to determine the structure of the sensor domain. Our research will provide new knowledge and contribute to building a comprehensive mechanistic understanding of stress kinases regulating translation. Our work will also contribute a new method, LRtcB RNA-seq, as a tool for studying mammalian mRNA decay.